Synthesis, molecular docking and antimicrobial activity of new fused pyrimidine and pyridine derivatives

Synthesis, molecular docking and antimicrobial activity of

new fused Pyrimidine and Pyridine derivatives

a,b, *

a*

a,c

Mohamed A. A. Radwan , Maha A. Alshubramy , Marwa Abdel-Motaal Bahaa A.

d

e

Hemdan , Dina S. El-Kady

a

Department of Chemistry, Collage of Science, Qassim University, Buraydah, Kingdom of

Saudi Arabia.

Department of Applied Organic Chemistry, National Research Center, Dokki, Egypt.

Department of Chemistry, Faculty of Science, El mansoura University, mansoura, Egypt

Environmental Microbiology Lab., Water Pollution Research Department, National Research

b

c

d

Centre, 33 El-Bohouth St., Dokki, Giza 12622, Egypt.

e

Hormones Department, National Research Centre, Dokki, Cairo, Egypt

*

Corresponding authors: Mohamed A. A. Radwan, Email: mrthaoan@qu.edu.sa or

m1radwan@yahoo.com; Maha A. Alshubramy, Email: maha.alshubramy@gmail.com;

Department of Chemistry, Collage of Science, Qassim University, Buraydah, Kingdom of

Saudi Arabia

Abstract: Synthesis of some new heterocyclic ring systems incorporated pyrimidine

and pyridine moieties starting from 1-(furan-2-yl)-3-(thiophen-2-yl) chalcone was

achieved. The structure of the new compounds was interpreted by spectral studies and

ESI-MS analysis. Antimicrobial investigations of the designated compounds were

performed towards some harmful pathogenic microbes. Antimicrobial tests proved that

compound 11 unveiled a greater antimicrobial activity than other designed compounds.

Docking of compound 11 into active site of DNA gyrase B chain displayed binding-

-1

o

energy of -13.05 kJ mol and distance at 3.18 A . Furthermore, docking investigation

was approved for the goal compounds into DNA gyrase B chain and exhibiting binding

-1

energy extended from-13.05 to -20.48 kJ mol .

Keywords: Pyrimidine, pyridine, chalcones, docking, DNA gyrase B chain,

antimicrobial.

1

. Introduction

Pyrimidine and pyridine ring systems are very important classes of compounds owing

to their wide-spectrum of biological activities. Nitrogen aromatic pyrimidine, pyridine,

and their analogs occur in nature and they show energetic role in the pitch of synthetic

heterocyclic chemistry [1]. Such heterocyclic compounds are extensively used for

many applications in medicinal science. Many of their derivatives are used as

antimicrobial [2-10], antiviral [11-13], antitumor [14-21] and anti-oxidant [22, 23]

agents in veterinary medicinal (Fig 1).

Fig.1

Chalcone derivatives have invited the attention of medicinal chemists due to their high

potential as chemical sources for designing and developing promising drugs [24, 25].

Some of the attracted chalcones were approved for clinical use, Metochalcone was

marketed as a choleretic drug [26, 27]. Hesperetin is a metabolite of hesperidin which

has better bioavailability, and as presented clinical trials of hesperidin methyl chalcone

for chronic venous lymphatic insufficiency and hesperidin trimethyl chalcone for trunk

or branch varicosis [28,29]. As well as sofalcone proved as an anti-ulcer agent that

improves the amount of mucosal prostaglandin [30, 31]. Also, Ro-09-0410 and its

prodrug, Ro-09-0415 were tested for rhinovirus infections [32] (Fig 2).

Fig.2

Furthermore, chalcone constituents are central initial ingredients for the synthesis of

unlike units of heterocyclic compounds, for example, pyrimidines, thiophenes, and

pyrazolines, etc. Several of these structures are extremely bioactive and are extensively

used as pharmaceuticals [33-36]. As part of our principal research program focused on

the synthesis of a variety of heterocycles molecules for biological evaluation [37-41].

herein, we report the synthesis of some heterocyclic ring systems incorporated with

pyrimidine and pyridine moieties starting from 1-(furan-2-yl)-3-(thiophen-2-yl)

chalcone. Antimicrobial investigations of the designated compounds were performed

towards some harmful pathogenic microbes. Furthermore, docking examination was

accomplished for the goal molecules to DNA gyrase B chain using MOE 2008.10

program.

1

. Results and Discussion

.1. Chemistry

1

Chalcone represent a significant synthetic intermediate candidate, mainly as a building

unit for the construction of several heterocyclic structures. Starting compound 1-(furan-

2

4

-yl)-3-(thiophen-2-yl) chalcone 4, was prepared as a previously reported method [42,

3] by the Claisen-Schmidt reaction of 2-actylfurane with 2-thiophenecaboxaldhyde in

the presence of catalytic KOH, Scheme 1.

Scheme 1

The actions of chalcone 4 towards some primary heteroaryl amines having NH group

2

connected at the α-site respect to the ring nitrogen (1,3-N, N-nucleophiles) was

considered. Consequently, chalcone 4 used for the synthesis of azolopyrimidines (10,

1

1 and 15), benzimidazothiazine 12 and pyrazolopyridine 13 compounds, Scheme 2.

Scheme 2.

Refluxing of chalcone 4 with 3-amino-1,2,4-triazole 5 in basic medium of potassium

hydroxide, afforded 5-(furan-2-yl)-7-(thiopen-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidine.

The construction of 10 was established based on spectroscopic methods IR, NMR, and

1

Mass. IR spectra not shown significant information. The H NMR spectrum is

characterized by the singlet-signals at δ 7.85 and 7.31 ppm of H-2 of the triazole ring

1

3

and H-6 for the pyrimidine respectively. Additionally, the C-NMR of compound 10

display thirteen carbons.

Also, chalcone 4 react with 5-amino-1,2,3,4-tetrazole monohydride 6 in basic condition

to

afford

4,7-dihydro-5-(furan-2-yl)-7-(thiopen-2-yl)[1,2,4,5]tetrazolo[1,5-a]

pyrimidine 11. The construction of compound 11 was established based on

spectroscopic tools; IR, NMR, and Mass. IR spectra presentation absorption band at

-1

1

3

160.30 cm distinguishing for NH proton. Also, the H-NMR data showed six

aromatic protons and two doublets, with coupling constant J=3.82Hz, at δ 5.1 and 6.6

ppm for 2H of pyrimidine and a single proton at 10.38 ppm assigned for the NH of

1

3

pyrimidine. Further, its C NMR spectrum showed four singles at δ 62.43, 102.57,

52.65 and 158.60 ppm which indicated to aliphatic carbon, C=C, and C=N in

1

pyrimidine ring, respectively. In addition to eight carbons of the furanyl and thienyl

rings (6CH & 2C ) appeared at 117.76, 120.88, 135.77, 136.18, 136.24, 136.52, 153.29,

q

and 155.28 ppm.

Reaction of cyclic thiourea, namely 2-mercaptobinzemidzole 7 with chalcone 4 afford

2

H-benzo[4,5]-imidazo[2,1-b][1,3]thiazine derivatives 12. The structure of this

compound was interpreted based on IR, NMR, and Mass. IR spectra have shown

3

155.40, 3111.66, 1623.44 and 1564.63 cm^-¹which indicated to aromatic-CH,

1

aliphatic-CH, C=N, and C=C respectively. Accordioning to H NMR spectrum two-

doublets, with J=8.16 Hz, at δ 6.44 and 6.65 ppm for thiazine protons, in addition to

the ten aromatic protons. Furthermore, the ¹³C NMR spectrum of compound 12

displaying eighteen carbons.

The reaction of 5-amino-1, 2-dihydro-3H-pyrazolo-3-one 8 with chalcone 4 in basic

condition only fused pyridine 13 was isolated. The product, 6-(furan-2-yl)-4-(thiopen-

2

-yl)-1H-pyrazolo[3,4-b]pyridine-3-ol 13 was interpreted based IR, NMR, and Mass

-1

tools. IR spectra have shown a broad peak at 3300-3000 cm which indicate the

1

occurred of OH group. Also, its H NMR displayed singlet at δ 6.7 ppm for pyridine

proton besides to six-aromatic protons and two signals at 10.53 and 11.43 ppm for -OH

1

3

and -NH. Additionally, the C NMR of 13 display distinguishing fourteen carbons.

The structure of possible pyrazolopyrimidine 14 was excluded as a result of the absence

of H-2 of pyrazole ring.

Furthermore, chalcone 4 react with 4-((4-nitrophenyl)diazenyl)-1H-pyrazole-3,5-

diamine afford one separated product. The product was recognized as 5-(furan-2-yl)-3-

((4-nitrophenyl)diazinyl)-7-(thiopen-2-yl)pyrazolo[1,5-a]pyrimidin-2-amine 15. IR of

1

5 viewing two bands owing to amino and nitro groups. Additionally, the H NMR of

1

3

compound 15 exposed a new singlet at δ 7.0 owing to pyrimidine-proton and C NMR

spectrum revealed eighteen carbons.

The behavior of chalcone 4 towards 3-cyanoacetyl indole 16 was considered. Thus, the

reaction of compound 4 with 16 in excess ammonium acetate and refluxing in acetic

acid afforded 6-(furan-2-yl)-2-(1H-indol-3-yl)-4-(thiophen-2-yl)nicotinonitrile 19. IR

of the product exposed two characteristic bands owing to NH and CN groups at 3389

-1

1

and 2204 cm respectively. Moreover, the H NMR of compound 19 display singlet at

1

3

δ 6.8 due to a proton of the construction of a new pyridine ring and C NMR spectrum

revealed twenty-two characteristic carbons.

Also, chalcone 4 react with ethylcyanoacetate or cyanoacetamide afford a single

product. Which was recognized as 6-(furan-2-yl)-4-(thiophen-2-yl-1,2-dihydro-2-oxo-

pyridine-3-carbonitrile 20. The spectral data of compound 20 was in a good agreement

with the literature values [44], Scheme 3.

Chlorination of compound 20 using POCl /PCl furnished 2-chloro-6-(furan-2-yl)-4-

3

5

(thiophen-2-yl) pyridine-3-carbonitrile 23. Chloropyridine 23 was established based on

IR, NMR, and Mass tools. IR of compound 23 exhibited characteristic absorption-band

-1

for (CN) at 2226 cm . As well as, the loss of C=O group of starting 2-oxo-pyridine-3-

1

carbonitrile 20. The H NMR 23 presented singlet at δ 6.72 (H-5 of pyridine), and its

+

mass displayed molecular ion peak at m/z (%): 286 (M , 5%).

Reaction of compound 20 with ethyl chloroacetate in basic medium at room

temperature afford Ethyl 2-((3-cyano-6-(furan-2-yl)-4-(thiophen-2-yl) pyridin-2-yl)

oxy) acetate 24. The structure of 24 was established based on IR, NMR, and Mass. IR

spectra of compound 24 showed two absorption bands distinguishing for (C≡N) at 2217

-1

1

cm and C=O group at 1749 cm . The H NMR of compound 24 showed new

characteristic triplet and quartet signals at δ 0.85 and 3.84 ppm for ethyl group and

1

3

singlet signal at δ 4.59 ppm for methylene protons. Its C NMR at δ 24.24 (Me), 71.29

CH ), 73.83 ppm (CH ) respectively. And its mass displayed molecular ion peak at

(

2

+

m/z: 335 (M 1).

Treatment of the chloropyridine derivative 23 with hydrazine in dioxane give 6-(furan-

-yl)-2-hydrazinyl-4-(thiophen-2-yl) pyridine-3-carbonitrile 25. The hydrazinyl

derivative 25 was established based on IR, NMR, and Mass. The IR spectra of

2

-1

hydrazinyl 25 showed characteristic bands for (CN) at 2201 cm and at 3203, 3160 for

1

(

NH-NH ). The H NMR of compound 25 disclosed new characteristic signals at δ 6.72

2

and 6.82 ppm (NH-NH ) group and singlet at δ 6.70 ppm for pyridine-proton. And its

2

+

mass disclosed molecular ion peak at m/z: 283 (M 1).

Furthermore, treatment of chloropyridine 23 with sodium azide afford 2-chloro-6-

(furan-2-yl)-3-(2H-tetrazol-5-yl)-4-(thiophen-2-yl) pyridine 27 instead of compound

2

6. The structure of 27 was established based on IR, NMR, and Mass. IR spectra of

-1

compound 27 showed absorption band distinguishing for (NH) at 3293 cm and loss

1

of CN group. H NMR of 27 exhibited a new characteristic broad signal at δ 6.58 ppm

1

3

(

NH) group. C NMR display a characteristic signal at δ 171.79 ppm (C-5 of new

+

construction tetrazole ring). And its mass showed molecular ion peak at m/z: 331 (M 2).

Ester 24 was treated with hydrazine to give 2-((3-cyano-6-( furan-2-yl)- 4-( thiophen-

2

-yl) pyridin-2-yl) oxy) acetohydrazid 28. The acetohydrazid derivative 28 was

interpreted based on IR, NMR, and Mass. IR spectra of 28 showed characteristic bands

-1

1

for (CN) at 2206 cm and at 1660 cm for (C=O). H NMR 28 disclosed characteristic

broad signals at δ 4.60 and 5.26 ppm (NH-NH ) group and singlet at δ 4.19 ppm for

2

1

3

methylene-protons (its C NMR at δ 73.93 ppm). And its mass disclosed molecular ion

+

peak at m/z: 322 (M -18).

Scheme 3.

Behavior of acetohydrazid 28 towards isatin and 4-nitrobenzaldehyde was considered

as outlined in scheme 4. The interpretations of the isolated compounds 31 and 32 were

an agreement with spectroscopic analysis; IR, NMR, and Mass (see experimental

section).

Scheme 4.

2

.2. In Silico Molecular Docking Screening

Modeling studies are essential to recognize the mechanisms of actions of drugs,

methods of interactions with designed compounds, and to integrate all trial indication

reported. Which are essential to find a reliable and exact image of biologically energetic

compounds. Thus, offer novel visions to plan novel medicinal compounds. Docking

investigation was approved for the goal synthetic molecules into DNA gyrase B chain

using MOE 2008.10 program. From the data gotten the new targets under investigation

displayed respectable fitting to the binding position of the protein surface and having

-1

binding energy extended from-13.05 to -20.48 kJ mol in contrast to the ligand

-1

(

Novobiocin complexes). Which displayed binding energy of-25.34 kJ mol , showed

arene- cation interaction between benzene ring of chromen moiety and Arg 75 and

formed five Hydrogen-bonds with the amino-acid residues; a) hydrogen of NH group

2

o

with Asp 72 in distance 2.75 A (36%); b) hydrogen of OH group with Asn 45 in

o

distance 2.78 A (68%); c) oxygen atom of C=O group formed two hydrogen bonds

o

with Arg 135 in distance 2.90 A (23%) and3..02 A (23%); d) hydrogen of OH group

o

with Asp 80 in distance 2.52 A (81%) (Fig 3).

Fig 3

-1

Compound 11 displayed binding-energy of -13.05 kJ mol and arene-cation contact

between thiophene and Arg 75 beside forming one H-bond with the amino acid

o

residues; a) hydrogen of NH group with Asp 72 in distance 3.18 A (11%) (Fig 4).

Fig 4

-1

Compound 25 displayed binding-energy of -17.18 kJ mol and arene-cation interface

between furan and Arg 75 beside forming four Hydrogen-bonds with the amino acid;

o

a) hydrogen of NH group with Asp 72 in distance 3.01A (14%) b) hydrogen of NH

2

o

group with Asp 72 in distance 2.69(70%) A c) nitrogen of NH group with Thr 166

2

o

and Gly 76 in distance 2.93(33%) and 3.01(33%) A respectively (Fig 5).

Fig 5

-1

Compound 27 revealed binding-energy of -20.48 kJ mol and arene-cation interaction

between furan and Arg 75 beside forming one H-bond with the amino acid; a) hydrogen

o

of NH group with Asp 72 in distance 2.77 A (33%) (Fig 6).

Fig 6

-1

Compound 28 exhibited binding-energy of -18.11 kJ mol and arene-cation interface

between thiophene and Arg 75 beside forming two H-bonds with the amino acid

o

residues; a) hydrogen of NH group with Asp 45 in distance 2.88 A (24%); b) nitrogen

2

o

of NH group with Tyr 94 in distance 2.97 A (18%) (Fig 7).

2

Fig 7

-1

Compound 31 unveiled binding-energy of -17.24 kJ mol and formed three H-bonds

with the amino acid residues; a) hydrogen of NH group with Asp 48 in distance 3.03

o

A (11%); b) nitrogen of hydrazine moiety and O-atom of C=O group with Lys 109 in

o

distance 2.60 A (43%) and 3.11 A (17%) (Fig 8).

Fig 8

Table 1

2

.3. Evaluation of antimicrobial activity

Data presented in Table 2 displayed that the diameters of inhibition zone of all tested

synthesized compounds against all tested strains, results found that all compounds were

indeed capable of halting microbial growth and have inhibition effects toward evaluated

microbial strains. Antimicrobial susceptibility tests proved that compound 11 unveiled

a greater antimicrobial activity than other compounds (Table 2).

The acquired results detected the highest diameters of the inhibition zone that was

caused by compound 11 were 18, 16, 15 and 13 mm for E. coli, L. monocytogenes, C.

albicans, and A. niger, respectively using the disc diffusion method, while by using

well diffusion assay the dimeters were 21, 18, 17, and 16 mm, respectively. Conversely,

the lowest antimicrobial effect was for compound 28, results exhibited that the zone of

inhibition diameters using disc diffusion assay toward E. coli, L. monocytogenes, C.

albicans, and A. niger were 5, 4, 4, and 3 mm, and 8, 7, 8 and 5 mm using well diffusion

assay. Furthermore, the experimental marks pointed out the diameter of the clear zone

using the well-diffusion method are wider than the disc diffusion method. Furthermore,

the investigational marks revealed that the tested Gram-negative bacterial species were

more sensitive than Gram-positive species to all tested synthesized compounds.

Furthermore, IC50% has been calculated for each compound as illustrated in Table 3

and some pictures of disc of the inhibition zone were demonstrated in Fig. 9.

Table 2

Table 3

Fig.9

Minimum inhibitory concentration

To assess the antimicrobial effectiveness and MIC values for each tested compound

against evaluated microbial lineages. Fig.10 illustrated the biocidal activity and MIC

values of three distinct concentrations of synthesized compound 11 against evaluated

species. The MIC value for inactivation of E.coli was 50 µg/mL within 30 min of time

intervals, while L. monocytogenes was taken 100 µg/mL at 5 min. MIC values for C.

albicans and A. niger were taken the same dose and contact time (100 µg/mL within 15

min).

Fig 10

Regarding compound 25, results demonstrated that 100 µg/mL within 15 and 30 min is

considered the effective dose toward E.coli and L. monocytogenes. Whereas MIC

values for C. albicans and A. niger were 150 µg/mL within 5 min (Fig. 11).

Fig 11

Data illustrated graphically in Figs. 12 and 13 pointed out that both compounds 27 and

2

8 have the same MIC values and inactivation rate against evaluated microbial lineages

and also were indeed able to prevent microbial growth. Thus, all four studied species

were susceptible to effective concentrations of compound 27 and 28, MIC values

toward all tested strains were 150 µg/mL within 5 min for E.coli and 15 min for the

other evaluated strains.

Fig 12

Fig 13

Results presented in Fig. 14 revealed that 150 µg/mL within 15 min of compound 31

were required to wholly inhibit bacterial growth of E.coli and L. monocytogenes, while

C. albicans and A. niger needed longer time (30 min)

Fig 14

2

.4. Structure activity relationship

In connection with the found activity values with the structural-moiety of the most

active designed molecules, it was approved that five applicants of the synthesized

compounds were the greatest activity. Antimicrobial activity of the synthesized

compounds was further investigated by the molecular docking approach, a method of

simulation of fitting ligands into binding site(s) of macromolecular targets. The marks

revealed that compound 11 with tetrazolopyrimidine substructure and with a binding

-1

o

energy of -13.05 kJ mol and distance at 3.18 A revealed higher antimicrobial activity

than other compounds. On the other hand, compound 25 with pyridine substructure

bearing 2-hydrazide and 3-carbonitrile groups which have binding-energy -17.18 kJ

-1

mol revealed good antimicrobial activity. Also, compound 27 incorporating pyridine

with tetrazole substituted ring at 3-position, in the present work, display more activity

than compound 31 and 28 respectively [2-6]. Compound 28 with acetohydrazid moiety

exposed less activity than the other compounds.

3

. Experimental

.1. General Considerations

All reagents and solvents were obtained from commercial suppliers and were used

without further purification. Microwave reactions were performed with a domestic

microwave oven Panasonic NN-SC688S, 120 V / 60 Hz, 1200W. Monitoring the

reaction and checking the purity of the final products were carried out by thin layer

chromatography (TLC) using silica gel precoated aluminum sheets (60 F254, Merck)

and visualization with Ultraviolet light (UV) at 365 and 254 nm. Melting point (°C)

were measured in open glass capillaries using stuart SMP30 advanced digital

apparatus and are uncorrected. Nuclear magnetic resonance spectra were recorded on

1

13

a Buker NMR spectrometer at 400.18 MHz for H and 100.62 MHz for C or on a

1

13

Buker Ascend 850 NMR spectrometer at 850.15 MHz for H and 213.77 MHz for C

Nuclear magnetic resonance center, KAU, Jeddah, KSA) or on a Buker Avance 500

(

1

13

NMR spectrometer at 500.13 MHz for H and 125.75 MHz for C at 25°C (Research

Unit, College of Pharmacy, Prince Sattam Bin Abdulaziz University, AlKharj, KAS).

The chemical shifts are expressed in (ppm) downfield from tetramethylsilane (TMS)

as internal standard; coupling constants (J) are expressed in Hz. Deuteriochloroform

(

CDCl ) and duteriodimethylsulphoxide (DMSO-d ) were used as solvents. The

3

6

splitting patterns were designated as: s (singlet), d (doublet), t (triplet), q (quarter) and

multiplet). Electrospray ionization mass spectra (ESI-MS) were recorded on Mass

Spectrometry Impact II™, Bruker (college of pharmacy, KAU, Jeddah, KSA).

(

3

.2. Chemistry

3

.2.1. Synthesis of 1-(furan-2-yl)-3-(thiophen-2-yl) prop-2-en-1-one chalcone.

Conventional method: KOH (0.16 gm, 3 mmol), was dissolved in H O (2.5 ml) and

2

EtOH (10 ml) at room temperature. (0.224 gm, 2 mmol) 2-thiophenecaboxaldhyde 1

was added to the solution. Then, 2-acetyl furan 2 (0.220 gm, 2 mmol) was added over

1

0 min. The mixture was stirred for 5 h at room temperature. A solid product was

poured into water (ice-cold) and neutralized with dil. HCl. After neutralization, the

solid was filtered, washed with cold EtOH, dried and recrystallized from ethanol to give

chalcone 4.

Microwave irradiation method: Repetition of the same scale reactions of the

conventional method. The reaction mixture was microwave irradiated for 4 min at 200

watts at 150°C. Completion of the reaction was identified by observing on TLC plates

(ethylacetate:petroleum ehther; 1:6, Rf: 0.3). After completion of the reaction was

treated similar to a conventional method to obtain chalcone 4. Mol. formula: C H O S,

1

8

2

conventional method yields 90%, microwave 86%, yellow crystals, m.p.74°C.

Characterization was the same as reported method [42, 43].

3

.2.2. General Method for the synthesis of compounds 10, 11, 12, 13, and 15.

A mixture of chalcone 4 (0.20 gm, 1 mmol) and the appropriate heterocyclic amine 5,

, 7, 8 and 9 (1.2 mmol) in DMF (10 ml) in the presence (26 mg, 0.1 mmol) of KOH

6

was refluxed for 0.45–2h at 110°C. The reaction monitored through TLC (petroleum

ether: ethyl acetate 8:2, Rf: 0.2, 0.3, 0.25, 0.3, 0.25, respectively), reaction mixture was

allowed to cool to ambient temperature, then poured into ice-water and the solid product

was collected by filtration followed by washing with ethanol. The crude product was

then recrystallized from appropriate solvent to give pure products 10, 11, 12, 13 and

1

5, respectively.

3

.2.2.1. 5-( Furan-2-yl)-7-(thiophen-2-yl) [1,2,4] triazolo[1,5-a] pyrimidine 10

Pink solid, (DMF); yield 85%, m.p. 150-153°C. IR (KBr, ν, cm-1): 3190- 3101(Ar-H),

1

706 (C=N), 1590 (C=C). H NMR (DMSO-d , δ ppm) 5.91 (dd, 1H, H-4, furan,

6

J=5.1Hz), 6.56 (dd, 1H, H-4, thiophen, J=9.35 Hz), 6.81 (dd, 1H, H-3, furan, J=3.4

Hz), 7.22 (dd, 1H, H-3, thiophen, J=2.2 Hz), 7.30 (dd, 1H, H-5, thiophen, J=6.0 Hz),

7

.31 (s, 1H, H-5, pyrimidine), 7.81 (dd, 1H, H-5, furan, J=5.01 Hz), 7.85 (s, 1H, H-3,

1

3

triazole); C NMR (DMSO d , δ ppm) 105.47 (CH, C-5, pyrimidine), 117.22 (CH, C-

6

C-3, furan), 118.30 (CH, C-4, furan), 132.32 (CH, C-4, thiophen), 133.90 (CH, C-3,

thiophen) , 137.35 (CH, C-5, thiophen), 139.55 (C, C-2, thiophen), 145.37 (CH, C-5,

furan) , 150.80 (C, C-2, pyrimidine), 154.97 (CH,C-3, triazole), 155.51 (C, C-2, furan)

,

159.50 (C, C-6, pyrimidine), 159.90 ( C,C-4, pyrimidine); ESI-MS, m/z: 269.0 [M+1];

Analysis Calcd. for C H N OS (268): C, 58.20; H, 3.01; N, 20.88; Found C, 58.32; H,

1

3

8

4

3

.08; N, 20.81.

.2.2.2. 5-( Furan-2-yl)-7-(thiophen-2-yl)-4,7-dihydrotetrazolo[1,5-a] pyrimidine 11

3

white solid (acetone); yield 80%, m.p. 244-246°C. IR (KBr, ν, cm-1): 3160 (NH), 3115-

1

3

5

086 (Ar-H), 1667 (C=N) 1610 (C=C); H NMR (DMSO-d , δ ppm) 5.15 (dd, 1H, H-

6

, pyrimidine, J=5.3 Hz), 6.31(dd, 1H, H-4, furan, J=6.73 Hz), 6.61 (d, 1H, H-4,

pyrimidine J=3.82 Hz), 6.71 (dd, 1H, H-4, thiophen, J=8.58 Hz), 6.75 (d, 1H, H-3,

furan, J=3.4 Hz), 6.94 (dd, 1H, H-3, thiophen, J=4.33 Hz), 7.24 (dd, 1H, H-5, thiophen,

J=4.59 Hz), 7.45(d, 1H, H-5, furan, J=1.36 Hz), 10.38 (1H, exchangeable with D O,

2

1

3

NH); C NMR (213 MHz, DMSO-d ) δ = 62.43 (CH, C-4, pyrimidine), 102.57 (CH,

6

C

C-5, pyrimidine), 117.76 (CH, C-4, furan), 120.88 (CH, C-3, furan), 135.77 (CH, C-

4

2

, thiophen), 136.18 (CH, C-3, thiophen), 136.24 (CH, C-5, thiophen), 136.52 (C, C-

, thiophen), 152.65 (C, C-6, pyrimidine), 153.29 (CH, C-5, furan), 155.28 (C, C-2,

+

furan), 158.60 (C, C-2, pyrimidine). ESI-MS, m/z: 274.2 [M+3], 271.5 [M ]; Analysis

Calcd. For C H N OS (271): C, 53.13; H, 3.34; N, 25.81; Found C, 53.22; H, 3.30; N,

1

2

9

5

2

5.74.

3

.2.2.3. 2-( Furan-2-yl)-4-(thiophen-2-yl) 2H-benzo [4,5] imidazolo[2,1-b] thiazine 12

yellow crystals (DMF) in yield 88%, m.p 230°C; IR (KBr, ν, cm-1): 3155-3111 (Ar-

1

H), 1623(C=N), 1564 (C=C); H NMR (DMSO d , δ ppm) 6.15 (dd, 1H, H-4, furan,

6

J=5.27 Hz), 6.44 (d, 1H, H-4, thiazine, J=8.16 Hz), 6.65 (dd, 1H, H-5, thiazine, J=8.16

Hz), 6.79 (dd, 1H, H-4, thiophen, J=8.67 Hz,), 6.88 (m, 3H, H-3, furan& 2H, Ar-H),

7

4

.16 (dd, 1H, H-3, thiophen J=4.76 Hz), 7.24 (d, dd, 1H, H-5, thiophen J=8.84 Hz),

1

3

.44 (dd, m, 3H, H-5, furan& 2H, Ar-H); C NMR (DMSO d , δ ppm) 112.27 (CH, C-

6

, thiazine), 113.44 (CH, C-6, benzimidazole) , 119.51 (CH, C-4, furan), 121.72 (CH,

C-3, furan), 121.83 (CH, C-9, benzimidazole), 123.27 (CH, C-5, thiazine), 124.98 (CH,

C-8, benzimidazole), 127.09 (CH, C-7, benzimidazole), 128.39 (CH, C-4, thiophen),

1

30.23(CH, C-3, thiophen), 131.46 (CH, C-5, thiophen), 131.74 (C, C-5,

benzimidazole), 132.98 (C, C-6, thiazine), 148.77 (C, C-2, thiophen), 149.41 (CH, C-

, furan), 150.85 (C, C-4, benzimidazole), 154.24 (C, C-2, furan), 173.34 (C, C-2,

5

+

thiazine). ESI-MS, m/z: 338.29 [M+2], 336.43 [M ]; Analysis Calcd. For

C H N OS : C, 64.26; H, 3.60; N, 8.33; Found C, 64.37; H, 3.39; N, 8.22.

1

8

12

2

3

.2.2.4. 6-(Furan-2-yl)-4-(thiopen-2-yl)-1H-pyrazolo[3,4-b]pyridine-3-ol 13

Black crystals (DMF); yield 67%, m.p. 288°C; IR (KBr, ν, cm-1): br. 3000- 3100 (OH

1

and NH), 1592 (C=N), 1544 ( C=C); H NMR (DMSO-d δ ppm) 5.91 (dd, 1H, H-4,

6

,

furan, J= 4.93 Hz),6.43 (d, 1H, H-4, thiophen, J= 3.23 Hz), 6.47 (dd, 1H, H-3, furan,

J=8.67 Hz), 6.71 (s,1H, H-5, pyridine), 7.00 (dd, 1H, H-3, thiophen, J=5.61Hz), 7.11

(d, 1H, H-5, thiophen, J=.68 Hz, 1H), 7.50 (dd, 1H, H-5, furan), 10.53 (s, 1H,

1

3

exchangeable with D O, OH), 11.43 (s, 1H, exchangeable with D O, NH); C NMR

2

(

DMSO d δ ppm) 105.38 (C, C-3, pyridine), 115.31 (CH, C-3, furan), 116.67 (CH, C-

6,

4

, furan), 119.05 (CH, C-5, pyridine), 135.13 (CH, C-4, thiophen), 135.49 (CH, C-3,

thiophen), 138.14 (CH, C-5, thiophen), 114.65 (C, C-4, pyridine), 114.93 (C, C-2,

thiophen), 151.31 (CH, C-5, furan), 154.99 (C, C-2, furan), 159.18(C, C-3, pyrazole),

1

3

5

3

60.61 (C, C-6, pyridine), 161.12 (C, C-2, pyridine). ESI-MS, m/z: 384.04 [M+1],

+

83.30 [M ]; Analysis Calcd. For C H N O S: C, 59.35; H, 3.20; N, 14.83; Found C,

1

4

9

3

2

9.77; H, 3.12; N, 14.49.

.2.2.5. 6-(Furan-2-yl)-3-((4-nitrophenyl)diazinyl)-7-(thiopen-2-yl)pyrazolo[1,5-

a]pyrimidin-2-amine 15

Red crystals; yield 60%, m.p. 270°C; IR (KBr, ν, cm-1): br. 3200 ( NH), 1690 ( C=N),

1

640 and 1440 (NO2), 1548 ( C=C); 1H NMR (DMSO-d δ ppm) (dd, 1H, H-4, furan,

6,

J=3.20 Hz), 6.79 (dd, 1H, H-4, thiophen, J=8.76 Hz), 6.85 (dd, 1H, H-3, furan, J=2.76

Hz), 7.00 (s,1H, H-5, pyrimidine), 7.03 (br. exchangeable with D O, NH ), 7.37(m, 4H,

2

Ar-H), 7.53 (dd, 1H, H-3, thiophen, J= 4.92 Hz), 7.68 (d, 1H, H-5, thiophen, J=8.48

1

3

Hz), 8.00 (dd, 1H, H-5, furan, J=3.12 Hz), C NMR (DMSO d δ ppm) 109.32 (CH,

6

,

C-5, pyrimidine), 121.04 (CH, C-3, furan), 121.32 (CH, C-4, furan), 124.83 (CH, C-4,

thiophen), 129.61 (2CH, C-2&C-6, benzene), 132.92 (2CH, C-3&C-5, benzene),

1

35.80 (CH, C-3, thiophen),136.63 (CH, C-5, thiophen), 137.54 (C, C-2, thiophen),

41.54 (C, C-4, pyrimidine), 141.72 (C, C-4, pyrazole), 143.54 (C, C-4, benzene),

47.25 (CH, C-5, furan), 153.85 (C, C-1, benzene), 154.21(C, C-2, pyrimidine), 156.01

(CH, C-2, furan), 159.76 (C, C-3, pyrazole), 165.25 (C, C-6, pyrimidine). ESI-MS,

+

m/z: 432.06 [M+1], 431.08 [M ]. Analysis Calcd. For C H N O S: C, 55.68; H, 3.04;

2

0

13

7

3

N, 22.73; Found C, 55.76; H, 3.01; N, 22.55.

.2.3. 6-(Furan-2-yl)-2-(1H-indol-3-yl)-4-(thiophen-2-yl)nicotinonitrile 19

3

A mixture of 3-cyanoacetylindole 16 (2 mmol, 0.368 gm) and ammonium acetate (8

mmol, 0.62 gm) in acetic acid AcOH (10 ml) was heated until complete dissolve. Then,

add chalcone 4 (2 mmol, 0.202 gm) and refluxed for 12 h. The reaction motoring by

TLC (ethylacetate:petroleum ehther; 1:6, Rf: 0.2). After the completion of reaction

allowed to cool at room temperature. The solid precepted was crystallized from AcOH

to afford the title compound as black crystals, yield 65 %, m.p. 236-238°C; IR (KBr, ν,

cm-1): 3389 (NH-Indole), 3150, 3098, 3081(CH-Ar), 2204 (CN), 1634, 1603 (C=N),

1

2

558(C=C); H NMR (DMSO d δ ppm) 5.90 (dd, 1H, H-4, furan, J=5.1 Hz), 6.35 (m,

6

,

H,Ar-H), 6.45 (dd, 1H, H-4, thiophen, J=8.67 Hz), 6.55 (dd, 1H, H-3, furan, J=3.4

Hz), 6.64 (dd, 1H, H-3, thiophen, J=7.48 Hz), 6.80 (s,1H, H-5, pyridine), 7.05 (m,

H,Ar-H), 7.13 (dd, 1H, H-5, thiophen, J=1.50 Hz), 7.51 (d, 1H, H-2, Indole, J=2.80

Hz), 7.54 (d, 1H, H-5, furan, J=7.65 Hz), 10.97 (s, exchangeable with D O, 1H, NH);

2

1

3

C NMR (DMSO-d δ ppm) 101.95 (C, C-3, pyridine), 115 (CH, C-3, benzene),

6

,

1

4

15.95 (CH, C-3, Indole), 116.13 (C, CN), 116.19 (CH, C-3, furan), 116.53 (CH, C-

, furan), 122.73 (CH, C-5, pyridine), 124.38 (CH, C-5, benzene), 125.21 (CH, C-6,

benzene),125.95 (CH, C-4, benzene), 129.95 (CH, C-4, thiophen), 132.10 (CH, C-3,

thiophen), 132.41 (CH, C-2, Indole), 133.55 (CH, C-5, thiophen), 133.84 (C, C-1,

benzene), 139.75 (C, C-2, thiophen), 140.64 (C, C-2, benzene), 149.60 (C, C-4,

pyridine), 150.21 (CH, C-5, furan), 153.53 (C, C-2, furan), 155.44 (C, C-2, pyridine),

+

1

61.73 (C, C-6, pyridine). ESI-MS, m/z: 368.07 [M+1], 367.42 [M ]. Analysis Calcd.

For C H N OS: C, 71.92; H, 3.57; N, 11.44; O; Found C, 71.92; H, 3.57; N, 11.44.

2

13

3

.2.2.1. 6-(Furan-2-yl)-4-(thiophen-2-yl-1,2-dihydro-2-oxo-pyridine-3-carbonitrile 20

A mixture of chalcone (10 mmol, 2.04 gm) and ethyl cyanoacetate 17 (1.13gm, 10

mmol) or cyanoacetamide 18 (2.10gm, 10 mmol) in n-butanol (50 ml) containing

ammonium acetate (6.16gm, 80 mmol) was heated under reflux for 3h or 5h

respectively. The reaction mixture was concentrated into its half volume and left to

cool. Then, the solid separated upon cooling was filtered, washed with water and

recrystallized from DMF to afford a compound 20 as yellow crystals, yield 60 % or

4

0% respectively, m.p. 300-303°C. The spectral data of compound was in good

agreement with the literature values[36].

3

.2.2.2. 2-Chloro-6-(furan-2-yl)-4-(thiophen-2-yl) pyridine-3-carbonitrile 23

A solution mixture of compound 20 (2.68 gm, 10 mmol), phosphorus oxychloride (10

ml, 0.10 mole) and phosphorous pentachloride (0.5 gm, 2 mmol) were fused on boiling

water bath for 10 h. After the reaction was completed, TLC (ethylacetate:petroleum

ehther; 1:6, Rf: 0.2)., the solution mixture was cooled and poured gradually into crushed

ice. The obtained precipitate was filtered off and dried and purified by silica gel column

chromatography with a gradient elution of ethyl acetate/n-hexane to afford 23. as

yellow crystals, yield 64% m.p. 163-165°C; IR (KBr, ν, cm-1): 3132, 2916 (CH-Ar),

1

2

226 (CN), 1603 (C=N), 1565 (C=C); H NMR (CDCl δ ppm) 6.08 (dd, 1H, H-4,

3

,

furan, J=5.1 Hz), 6.70 (dd, 1H, H-4, thiophen, J=7.8 Hz), 6.72 (s,1H,H-5, pyridine),

.78 (dd, 1H, H-3, furan, J=4.08 Hz), 7.07 (dd, 1H, H-3, thiophen, J=6.12 Hz), 7.09

6

1

3

(

dd, 1H, H-5, thiophen, J=2.21 Hz), 7.45 (dd, 1H, H-5, furan, J=1.10 Hz); C NMR

(

CDCl δ ppm) 110.37 (C, C-3, pyridine), 120.10 (CH, C-3, furan), 120.90 (C, CN),

3

,

1

22.09 (CH, C-4, furan), 122.81 (CH, C-5, pyridine), 136.00 (CH, C-4, thiophen),

37.24 (CH, C-3, thiophen), 137.30 (CH, C-5, thiophen), 143.54 (C, C-2, thiophen),

52.74 (C, C-4, pyridine), 154.93 (CH, C-4, furan), 157.97 (C, C-2, pyridine), 158.14

+

(

C, C-2, furan), 161.88 (C, C-6, pyridine). ESI-MS, m/z: 286 [M ]. Analysis Calcd.

For C H ClN OS: C, 58.64; H, 2.46; Cl, 12.36; N, 9.77; Found C, 58.78; H, 2.41; N,

1

4

7

2

9

.54.

.2.2.3. 6-(Furan-2-yl)-2-hydrazinyl-4-(thiophen-2-yl) pyridine-3-carbonitrile 25

3

A mixture of the chloropyridine derivative 23 (2.86 g mmol) and hydrazine hydrate

0% (1.57 ml, 50 mmol) in dioxane (10 ml) was heated under reflux for 12 h, TLC,

ethylacetate:petroleum ehther; 1:6, Rf: 0.3). (ethylacetate:petroleum ehther; 1:6, Rf:

.3). The formed precipitate was filtered off, dried and recrystallized from methanol to

8

(

0

give the hydrazine derivative 25 as yellow crystals, yield 64% m.p. 200°C; IR (KBr, ν,

cm-1): 3203, 3160 (NH-NH ), 2974 (CH-Ar), 2201 (CN), 1600 (C=N), 1572 (C=C);

2

1

H NMR (CDCl δ ppm) 6.00 (dd, 1H, H-4, furan, J=5.2 Hz), 6.54 (dd, 1H, H-4,

3

,

thiophen, J=8.68 Hz, 1H), 6.61 (dd, 1H, H-3, furan, J=3.32 Hz, 1H), 6,70 (s,1H,H-5,

pyridine), 6.72 (s, exchangeable with D O, 2H, NH ), 6.82 (s, exchangeable with D O,

2

H, NH), 6.98 (m, 1H, H-3, thiophen), 7.14(m, 1H, H-5, thiophen, 7.90 (m, 1H, H-5,

1

3

furan, J=1.12 Hz); C NMR (CDCl δ ppm) 111.77 (C, C-3, pyridine), 120.66 (CH,

3

,

C-3, furan), .120.93 (C, CN), 121.10 (CH, C-4, furan), 123.04 (CH, C-5, pyridine),

39.16 (CH, C-4, thiophen), 139.132 (CH, C-3, thiophen), 139.99 (CH, C-5, thiophen),

1

48.40 (C, C-2, thiophen), 155.27 (C, C-4, pyridine), 156.74 (CH, C-5, furan), 158.03

(C, C-2, pyridine), 163.21 (C, C-2, furan), 163.50 (C, C-6, pyridine). ESI-MS, m/z:

+

2

1

3

83.05 [M+1], 282.32 [M ]. Analysis Calcd. For C H N OS: C, 59.56; H, 3.57; N,

1

4

10

4

9.85; Found C, 59.73; H, 3.51; N, 19.76.

.2.2.4. 2-Chloro-6-(furan-2-yl)-3-(2H-tetrazol-5-yl)-4-(thiophen-2-yl)pyridine 27

A mixture of the chloropyridine 23 (0.286 gm, 1 mmol) in 10 ml of DMF and then add

0.195 gm, 3 mmol) of sodium azide in 5 ml of water was refluxed for 8 h, After the

(

reaction was completed, TLC, (ethylacetate:petroleum ehther; 1:6, Rf: 0.3), the

precipitate was removed by filtration and filtrate poured into water (ice-cold) and

neutralized with dil. AcOH. After neutralization, the solid was filtered, washed with

water, crystallized from ethanol to give 27 as a red powder in a yield (53%) m.p. 160

°

C. IR (KBr, ν, cm-1): 3293 (NH), 3114, 3112 (CH-Ar), 1647 (N=N), 1597 (C=N),

547,1539 (C=C), 703(C-Cl); 1H NMR (DMSO-d δ ppm) 6.25 (dd, 1H, H-4, furan,

1

6

,

J=5.2 Hz), 6.58 (br, exchangeable with D O, 1H, NH), 6.70 (s,1H,H-5, pyridine), 6.77

2

(dd, 1H, H-4, thiophen, J=3.4 Hz), 6.84 (dd, 1H, H-3, furan, J= 8.8 Hz), 7.38 (dd, 1H,

H-3, thiophen, J=4.8 Hz), 7.38 (dd, 1H, H-5, thiophen, J=6.15 Hz), 7.45 (m, 1H, H-5,

1

3

furan, J=1.4 Hz); C NMR (DMSO-d δ ppm) 116.28 (CH, C-3, furan), 122.43 (CH,

6

,

C-4, furan), 123.18 (CH, C-5, pyridine), 127.76 (CH, C-4, thiophen), 139.01 (CH,

C-3, thiophen), 139.29 (CH, C-5, thiophen), 139.59 (C, C-3, pyridine), 140.21 (C, C-

2

, thiophen), 148.02 (C, C-2, pyridine), 155.44 (C, C-4, pyridine), 156.66 (CH, C-5,

furan), 160.96 (C, C-2, furan), 162.36 (C, C-6, pyridine), 171.79 (C, C-5, tetrazole).

+

ESI-MS, m/z: 329 [M ]. Analysis Calcd. For C H ClN OS: C, 50.99; H, 2.45; Cl,

1

4

8

5

1

0.75; N, 21.24; Found: C, 51.08; H, 2.41; N, 21.19.

3

.2.2.5. Ethyl 2-((3-cyano-6-(furan-2-yl)-4-( thiophen-2-yl) pyridin-2-yl) oxy) acetate

2

4

A mixture of 20 (2.68gm, 10 mmol) and (1.52 gm, 11 mmol) potassium carbonate was

stirred in dry DMF (20 ml) for 1h, followed by the addition of ethyl chloroacetate (1.44

gm, 10 mmol), the reaction mixture was stirred at room temperature for 24 h., then

poured into ice-water and acidified with HCl. to give the crude product which purified

by silica gel column chromatography with a gradient eluent of ethyl acetate/ petroleum

ether to afford 24 as yellow crystals, yield: 95%; m.p.124 °C. IR (KBr, ν, cm-1): 2976

1

(

CH-Ar), 2217 (CN), 1749 (C=O), 1599 (C=N), 1547, 1539 (C=C); H NMR (CDCl

3

,

δ ppm) 0.85 (t, 3H, Me, J=19.04 Hz), 3.84 (q, 2H, CH , J= 21.33 Hz), 4.59 (s, 2H,

2

CH ), 6.15 (dd, 1H, H-4, furan, J=5.1 Hz), 6.69 (dd, 1H, H-3, furan, J=3.99 Hz), 6.81

2

(dd, 1H, H-4, thiophen, J=8.84 Hz), 6.85 (s,1H, H-5, pyridine), 7.16 (dd 1H, H-3,

thiophen, J=6.12 Hz), 7.18 (dd, 1H, H-5, thiophen, J=2.29 Hz), 7.57 (dd, 1H, H-5,

1

3

furan, J=6.8 Hz, 1H); C NMR (CDCl , δ ppm) 24.24 (Me), 71.29 (CH ), 73.83 (CH ),

3

2

1

00.16 (C, C-3, pyridine), 120.82 (CH, C-4, furan), 122.54 (C, C-CN), 122.69 (CH,

C-4, furan), 125.62 (CH, C-5, pyridine), 138.73 (CH, C-4, thiophen), 139.43 (CH,

C-3, thiophen), 139.76 (CH, C-5, thiophen), 147.38 (C, C-2, thiophen), 155.00 (C, C-

4

, pyridine), 158.37 (CH, C-5, furan), 158.93 (C, C-2, furan), 162.09 (C, C-6,

pyridine), 174.05 (C, C-2, pyridine), 178.23 (C=O). ESI-MS, m/z: 355.06 [M+1],

3

6

53.25 [M-1]. Analysis Calcd. For C H N O S: C, 61.01; H, 3.98; N, 7.91; Found: C,

18 14 2 4

1.22; H, 3.92; N, 7.86.

3

.2.2.6. 2-((3-Cyano-6-( furan-2-yl)- 4-( thiophen-2-yl) pyridin-2-yl) oxy)

acetohydrazid 28

The compound 24 (3.54 gm, 10 mmol) was added to hydrazine hydrate 80% (20 mmol,

0

.627 ml) in absolute ethanol (20 ml) and refluxed for 7 h, the reaction monitoring by

TLC (ethylacetate:petroleum ehther; 1:6, Rf: 0.24). After finishing concentrated the

reaction mixture was and cooling, the precipitate obtained was filtered off, crystallized

from ethanol to give 28 as white crystals, yield: 95%; m.p. 245°C; IR (KBr, ν, cm-1):

1

3

132, 2916 (CH-Ar), 2206 (CN), 1660 (C=O), 1603 (C=N), 1565 (C=C); H NMR

(DMSO-d δ ppm) 4.19 (s, 2H, CH ), 4.60 (s, exchangeable with D O, 2H, NH ), 5,26

6, 2 2 2

(

s, exchangeable with D O, H, NH), 5.87 (dd, 1H, H-4, furan, J= 4.85 Hz), 6.44 (m,

2

H, H-4, thiophen, H-3, furan), 6.71 (s,1H, H-5, pyridine), 7.08 (m, 3H, H-3& H-5

1

3

thiophen, H-5, furan,); C NMR (DMSO-d δ ppm) 73.93 (CH ), 99.42 (CH, C-3,

6

,

2

pyridine), 120.30 (CH, C-3, furan), 123.20 (C, CN), 123.85 (CH, C-4, furan), 125.42

(

CH, C-5, pyridine), 139.05 (CH, C-4, thiophen), 140.53 (CH, C-3, thiophen), 141.42

CH, C-5, thiophen), 146.35 (C, C-2, thiophen), 156.92 (C, C-4, pyridine), 158.07 (CH,

(

C-5, furan), 158.64 (C, C-2, furan), 161.11 (C, C-6, pyridine), 173.39 (C, C-2,

pyridine), 176.98 (C=O). ESI-MS, m/z: 341.36 [M+1]. Analysis Calcd. For

C H N O S: C, 56.46; H, 3.55; N, 16.46; Found : C, 56.55; H, 3.32; N.

1

6

12

4

3

.2.3.

2-((3-Cyano-6-(furan-2-yl)-4-(thiophen-2-yl)pyridin-2-yl)oxy)-N'-(2-

oxoindolin-3-ylidene)acetohydrazide 31

A mixture of the compound 28 (0.34 gm, 1 mmol) and isatin 29 (0.147 gm, 1 mmol) in

2

0 ml ethanol containing drops of acetic acid was refluxed at the water bath. The

reactions motoring by TLC (ethylacetate:n-hexane; 2:5, Rf: 0.24). After the completion

of reactions, allowed to cool at room temperature. then left overnight at room

temperature. The formed precipitate was filtered, dried and recrystallized from

methanol to give the title compound 32 as yellow crystals, yield: 96%; m.p.246 °C; IR

(KBr, ν, cm-1): 3100- 3300 (br. 2NH), 2226 (CN), 1705, 1715 (2C=O), 1603 (C=N),

1

565 (C=C); H NMR (DMSO-d δ ppm) 5.22 (s, 2H, OCH CO), 6.26 (dd, 1H, H-4,

6

,

2

furan, J=5.2 Hz), 6.54 (d, H-4, thiophene, J=7.84 Hz), 6.68 (t, 1H, H-4, benzene,

J=15.28 Hz, 1H), 6.83 (m, 1H, H-3, furan), 6,96 (dd, 1H, H-3, thiophene, J=8.84 Hz),

7

.02 (t, 1H, H-5, benzene, J=15.32 Hz), 7.22 (s,1H, H-5, pyridine), 7.51 (m, 1H, H-5,

thiophene J=0.92 Hz), 7.69 (m, 2H, H-3& H-5, benzene), 7.77 (d, 1H, H-5, furan,

J=7.68 Hz), 10.74 (s, exchangeable with D O, NH), 11.18 (s, exchangeable with D O,

2

1

3

NH); C NMR (CDCl ) 89.99 (CH , OCH CO), 110,98 (C, C-3 pyridine), 111.47 (CH,

3

2

C-3, furan), 113.74 (CH, C-5, pyridine), 114.12 (CH, C-4, furan), 115.99 (C, CN),

1

16.33 (C,C-2, benzene), 122.54 (CH, C-6, benzene), 127.21 (CH, C-4, benzene),

29.65 (CH, C-4, thiophene), 131.17 (2CH, C-3, thiophene & C-3, benzene), 131.99

(

2CH, C-5, thiophene& C-5, benzene), 133.68 (C,C-2, thiophene), 137.12 (C, C-1,

benzene), 144.85 (CH,CH=N), 147.24 (CH, C-5, furan), 148.77 (C, C-4, pyridine) ,

1

49.30 (C, C-2, furan), 151.81 (C, C-6, pyridine), 164.55 (C, C-2, pyridine), 164.55

+

(

C, C=O), 168.81 (C, C=O). ESI-MS, m/z: 470.06 [M+1], 469.48 [M ]. Analysis

Calcd. For C H N O S: C, 61.40; H, 3.22; N, 14.92; Found: C, 61.47; H, 3.13; N,

2

4

15

5

4

1

4.87.

3

.2.4.

2-((3-Cyano-6-(furan-2-yl)-4-(thiophen-2-yl)pyridin-2-yl)oxy)-N'-(4-

nitrobenzylidene)acetohydrazide 32

A mixture of the compound 27 (0.34 g, 1 mmol) and 4-nitrobenzaldehyde 30 (1 mmol)

in absolute ethanol (20 ml) was refluxed at the water bath. The reactions motoring by

TLC (ethylacetate:n-hexane; 1:3, Rf: 0.36).. After the completion of reactions allowed

to cool at room temperature. then left overnight at room temperature. The formed

precipitate was filtered, dried and recrystallized from DMF to give the title compound.

(DMF) Whit crystals, yield: 96%; m.p. 273°C.; IR (KBr, ν, cm-1): 3300 (NH), 2221

1

(

CN), 1695 (C=O), 1603 (C=N), 1550 and 1400 (NO ), 1565 (C=C); H NMR (DMSO

2

d δ ppm) 5.21 (s, 2H, OCH CO), 6.26 (dd, 1H, H-4, furan, J=5.2 Hz), 6.75 (d, 1H,

6

,

2

H-4, thiophene, J=3.28 Hz), 6.93 (m, 1H, H-3, furan),, 7.18 (s,1H, H-5, pyridine), 7.57

(m, 5H, H-2, H-6,H-3& H-5 benzene& H-3, thiophene), 7.57 (s,1H, N=CH), 7.75 (m,

1

3

1

6

H, H-5, furan), 11.56 (s, exchangeable with D O, NH); C NMR (CDCl δ ppm)

2 3,

3.71(OCH CO), 89.20 (C, C-3, pyridine), 110.06 (CH, C-3, furan), 112.97 (CH, C-5,

2

pyridine), 113.17 (CH, C-4, furan), 115.45 (C, CN), 124.04 (2CH, C-2& C-6,

benzene), 127.89 (2CH, C-3& C-5, benzene), 128.85 (CH, C-4, thiophene), 130.32

(CH, C-3, thiophene), 131.14 (CH, C-5, thiophene), 136.34 (C, C-2, thiophene), 140.18

(C, C-1, benzene), 141.72 (C, C-4, pyridine), 146.46 130.31 (C, C-5, furan), 147.81

(CH, CH=N), 147.92 (C, C-4, benzene), 148.51(C, C-2, furan), 151.08 (C, C-6,

pyridine), 164.26 (C, C-2, pyridine), 168.76 (C=O) ESI-MS, m/z: 474.06 [M+1],

+

4

1

3

73.34 [M ], 472.33[M-1]. Analysis Calcd. For C H N O S: C, 58.35; H, 3.19; N,

2

3

15

5

4.79; Found: C, 58.49; H, 3.11; N, 14.72.

.3. In Silico Molecular Docking Screenings.

Docking estimation of the most active antibacterial compounds 11, 25, 27, 28 and 31

was achieved by Molecular Operating Environment (MOE) 2008.10 releases of

Chemical Computing Group, Montreal, Canada (http://www. chemcomp. com.). The

program operated on an Intel(R) Core (TM) i3-32100 CPU@3.10GHz 3.09 GHz

processor, 3.41 GB of RAM, Microsoft Windows XP.

Docking was done against the active site of the protein molecular surface of DNA

gyrase B chain (PDB ID: 1KIJ) in complex with Novobiocin (was downloaded from a

protein data bank (http://www. rcsb. org/-pdb) (PDB ID: 1KIJ) [45].

The DNA gyrase enzyme has a crucial role in bacterial cell viability [46]. DNA gyrase

B) possesses the ATPase activity, providing a sufficient amount of energy for the DNA

supercoiling [47] DNA gyrase remains an attractive target due to its presence across all

microbes [48]. Targeting DNA gyrase with an inhibitor is a new strategy for dealing

with antimicrobial resistance by disrupting DNA synthesis, leading to cell death and

reducing the development of resistance [49].

The protein crystal construction was prepared for docking via removing of water

molecules, addition and elimination of polar hydrogen atoms then separation of the

active pocket. The active site was measured to be the site where co-crystalline ligand

namely, Novobiocin complexes (PDB ID: 1KIJ). The crystal structure of Gyrase B 43K

ATPase domain in complex with novobiocin, is one of the most potent inhibitors of

gyrase showing large conformational changes of the subdomains within the dimer [50].

The co-crystalline ligand was re-docked in the active pocket to ensure the docking

process was effective and the active pocket was saved to be used for docking simulation

of the designated new compounds.

The building of the selected compounds for docking was drawn in Chem Draw Ultra

1

0.0 (ChemOffice package) and saved. Before the molecular docking, preparation steps

must be done as follow; a) converting the 2D structure of ligands to their 3D form; b)

addition and removing of polar hydrogen atoms; c) energy minimized using the

MMFF94x force field until a RMSD (Root-mean-square deviation) of atomic position

-1

gradient of 0.01 Kcal mol Å was gotten and saved as MOE. MMFF94x was reported

as an efficient force field for minimizing ligand-protein complexes [51].

The docking Algorithm was done by MOE-DOCK default. It uses a flexible, rigid

technique for posing the molecule inside the cavity. All rotatable bonds of ligands are

allowed to undergo free rotation to be placed into the rigid receptor binding site. The

docking scores were expressed in negative energy terms; the lower the binding free

energy, the better the binding affinity [52], and the ligand interactions (hydrogen

bonding and hydrophobic interaction) with DNA gyrase B chain was determined [53].

3

.3.1. Evaluation of antimicrobial activity.

.3.2. Preparation of working stock solution

By dissolving 500 mg of each synthesized compound in 2 ml DMSO, stock solutions

of five synthetic compounds (11, 25, 27, 28 and 31) were prepared and ready to be used.

Four prospective pathogenic microbes were examined against three distinct

concentrations (50, 100 and 150 μg/mL) of each synthesized compound.

3

.3.2. Microorganisms used and preparation

Four microbial species used in the present study were as follows: Escherichia coli

ATCC 25922 as an example for Gram-negative bacteria, Listeria monocytogenes

ATCC 25152 as an example for Gram-positive bacteria, Candida albicans ATCC

1

0231 as an example for yeast; and Aspergillus niger ATCC 6275 as an example for

fungi. All bacterial and fungal species were cultured into Tryptic Soya broth and Malt

o

extract broth for incubation overnight at 37 C , and harvested in the mid-log phase. For

2

0 minutes, each culture tube (falcon 50 ml) was centrifuged at 4000 rpm. The

supernatant was discarded and the pellet cells were taken and washed three times to

remove any undesired particulates using phosphate-buffered saline. Final cells

6

concentration of culture suspension was 2.5x10 CFU/mL [54].

3

.3.3. Antimicrobial susceptibility testing

a. Kirby–Bauer test (well and disc diffusion)

Well and disc diffusion assays were performed by disseminating 100μL on the surface

of Müller Hinton agar (MHA) (BBLTM, Germany) plates from each prospective

microbial pathogen culture. For well diffusion assay, the wells (6 mm in diameter) were

punctured with such a sterile driller upon the surface of MHA media and 100 μL of

each antiseptic solution studied was inserted into each well [55]. Whilst the sterile filter

paper discs (6 mm in diameter) were saturated with 100 μL of the tested antiseptic and

placing the discs onto the surface of MHA plates. After appropriate incubation, the

diameters of clear zones were measured in mm using a scale.

b. Minimum inhibitory concentrations (MIC)

The MIC values of each synthesized compound were studied against the target

microbial pathogens according to Gharib et al [56]. In brief, 100 μL of each fresh

microbial culture was injected into 10 mL sterile distilled water tubes that containing

different concentrations (50, 100 and 150 μg/mL) of tested antiseptics. While control

tubes for each tested strain were free from disinfectants. The specific conditions of all

experiments were at room temperature and shaking at 250 rpm. Samples were taken

from each tube with four different time intervals (5, 15, and 30 min) for counting of the

viable bacterial cells. The densities of living cells before (initial count) and after

exposure to synthesized compounds were counted using the pour plate method. All

experiments were performed in triplicate and repeated at least twice on different days

[57].

Author Contributions: Radwan .M. A. A. conceived the research project, participated in all

steps of the research, interpreted the results, discussed the experimental data and prepared the

manuscript; Maha A. Alshubramy do the experimental part and, participated in all steps of the

manuscript. Bahaa A. Hemdan prepared antimicrobial work. Dina S. El-Kady makes docking

studies. All authors read, discussed and approved the manuscript.

Acknowledgment:

Authors are gratefully acknowledged Deanship of Graduate Studies and department of

chemistry collage of science, Qassim University for support this project.

Conﬂict of interest

The authors declare no conﬂict of interest.

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CH₃

N

O S O

O

CH₃

EtO C

H NSCHNHN

2

N

O

N

H

N

H N

N

H

N

H

2

O

HN

N

H

N

H

N

Cl

(

C) Slidenafil

(A)

(B)

Fig.1 Examples of bioactive fused Pyrimidine and Pyridine compounds

Fig.2 Some chalcones that have been marketed or clinically tested

Scheme 2 Reagents and conditions: (i) KOH, EtOH, stirring 5 h.

Scheme 3 Reagents and conditions: (i) KOH, DMF, reflux.

O

S

4

NC

(ii)

(i)

O

NC

^N^H2

OEt

N

17

H

18

16

S

CN

O

CN

N

H

O

20

N

H

19

O

(iii)

Cl

POCl /PCl₅

(iv)

3

OEt

22

S

CN

O

OEt

N

Cl

N

O

23

24

(

vii) N H . H O

2

4

2

NaN₃(vi)

(v) N H . H O

2 4 2

S

CN

NHNH₂

S

N

HN

CN

O

N

S

^N^H^N^H2

O

25

N

Cl

CN

N₃

O

28

O

27

N

O

26

Scheme 3 Reagents and conditions: (i) NH OAc, AcOH, reflux 12 h. (ii) NH OAc, n-

4

butanol reflux 3h/ 5h. (iii) fusion at 80, 10 h. (iv) K CO , DMF, stirring, 24 h. (v)

2

3

dioxane, reflux 12 h. (vi) H O, DMF, reflux 8 h. (vii) EtOH, reflux 7 h.

2

Article Doi

Synthesis, molecular docking and antimicrobial activity of new fused pyrimidine and pyridine derivatives

DOI: 10.1016/j.bioorg.2019.103516

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bioorg.2019.103516

Full text of DOI:10.1016/j.bioorg.2019.103516

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